colony-stimulating factors - Europe PMC

Proc. Natl. Acad. Sci. USA

Vol. 88, pp. 6239-6243, July 1991 Medical Sciences

Direct proliferative actions of stem cell factor on murine bone marrow cells in vitro: Effects of combination with colony-stimulating factors (hemopoietic colonies/autoradiography/clonal cultures/clone transfer)

D. METCALF* AND N. A. NICOLA The Walter and Eliza Hall Institute of Medical Research, P.O. Royal Melbourne Hospital 3050,

Victoria, Australia

Contributed by D. Metcalf, April 16, 1991

Stem cell factor (SCF), the ligand for the c-kit ABSTRACT protooncogene product, was able to stimulate blast cell and granulocytic colony formation by precursors from normal murine bone marrow. The blast cell colonies contained a high content of progenitor cells able to form macrophage and/or granulocyte colonies. Clone transfer studies, the secondary culture of colony cells, and the culture of populations freed of accessory cells all indicated a direct proliferative action of SCF. SCF receptors were present in high numbers on blast cells and in lower numbers on immature granulocytic, monocytic, and eosinophilic cells. Combination of SCF with granulocyte, granulocyte-macrophage, or multipotential colony-stimulating factors, but not macrophage colony-stimulating factor, resulted in enhancement of colony size. Granulocyte colony-stimulating factor enhanced cell proliferation initiated by SCF, but not vice-versa, and resulted in a 10-fold increase in colony cell numbers and a 7-fold increase in progenitor cells in blast colonies. No evidence was obtained that SCF, alone or in combination with granulocyte colony-stimulating factor, could stimulate self-generation by blast colony-forming cells.

The protooncogene c-kit encodes a membrane receptor, present on hemopoietic and other cells but defective or absent in the various W (dominant spotting) mutant mice with defective stem cells and erythropoiesis (1, 2). The ligand for the c-kit product has recently been purified and cloned (3, 4), and production of this molecule is absent or defective in mice with the Steel mutation that also exhibit defective erythropoiesis. Initial studies documented the ability of the c-kit ligand [termed stem cell factor (SCF) or mast cell growth factor] to stimulate the proliferation of mast cells (3, 4) and primitive hemopoietic precursor cells (3) and to enhance erythropoietin-stimulated erythropoiesis and granulocytemacrophage colony formation in the presence of colonystimulating factors (4, 5). The present studies were undertaken to establish whether or not SCF is a direct proliferative stimulus for hemopoietic cells and the nature of the enhanced cell proliferation observable when SCF is used in combination with the various

colony-stimulating factors.

for up to 7 days in a fully humidified atmosphere of 10%o CO2 in air. The transfer of intact clones to recipient cultures or the reculture of resuspended colony cells was performed as described (6). Colony formation was scored by using a dissection microscope at 35x magnifications, and cultures were then fixed by using 1 ml of 2.5% glutaraldehyde. The intact cultures were floated onto slides and, after drying, were stained for acetylcholinesterase and then stained with Luxol fast blue and hematoxylin. Stimuli. All stimuli used were recombinant nonglycosylated factors purified after expression in Escherichia coli: SCF (rat) and granulocyte colony-stimulating factor (G-CSF; human 108 units/mg) respectively supplied by K. Zsebo and L. Souza (Amgen Biologicals); and recombinant murine granulocyte-macrophage colony-stimulating factor (GMCSF), macrophage colony-stimulating factor (M-CSF), and multipotential colony-stimulating factor (Multi-CSF) produced in this laboratory. After purification, the latter group of CSFs assayed at 3 x 108, 1 x 108, and 1 x 108 units/mg of protein, respectively, by the methods for unit estimation described (6). Stem Cell Enrichment. Enrichment by fluorescenceactivated cell sorting (FACS) of stem and progenitor cells from normal mouse bone marrow was based on methods described previously (7). Autoradiographic Studies. Recombinant rat SCF was radiolabeled with 1251 (125I-SCF) by using iodine monochloride (8) to a specific radioactivity of 30,000 cpm/ng. BALB/c bone marrow cells (5 x 106) were incubated with 125I-SCF (200,000 cpm) for 45 min at 230C in 60 1.l of RPMI 1640 medium containing 10%o fetal calf serum and 10 mM Hepes buffer (pH 7.3). Specificity was determined in parallel tubes containing 300 ng of unlabeled SCF (3400 + 100 cpm versus 350 + 40 cpm). Cytocentrifuge cell preparations were fixed with 2.5% glutaraldehyde and then exposed for 21 days with Kodak NTB2 emulsion. After development, the preparations were stained with May-Grunewald Giemsa.

RESULTS SCF, acting alone in cultures of 75,000 bone marrow cells, stimulated colony formation, but the concentration required was 1000-fold higher than for the colony-stimulating factors (Fig. 1). The maximum number of colonies developing was only two-thirds of that stimulated by GM-CSF but twice the number stimulated by G-CSF. After 7 days of culture, SCF-stimulated colonies had a distinctive morphology. The

MATERIALS AND METHODS Cultures. Primary cultures were performed in 35-mm Petri dishes usually containing 75,000 bone marrow cells from 8-week-old C57BL/6/WEHI mice in a 1-ml volume of Dulbecco's modified Eagle's medium with a final concentration of 20% (vol/vol) fetal calf serum and 0.3% agar (6). Stimuli in a volume of 0.1 ml were added prior to the addition of the cells in agar-medium. After gelling, cultures were incubated

Abbreviations: SCF, stem cell factor; CSF, colony-stimulating factor; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; Multi-CSF, multipotential colonystimulating factor; IL-4, IL-5, and IL-6, interleukins 4, 5, and 6; FACS, fluorescence-activated cell sorting. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 88 (1991) 800

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Concentration ng/rrd FIG. 1. Stimulation of colony formation by SCF, G-CSF, or GM-CSF in cultures of 75,000 C57BL/6/WEHI bone marrow cells. Cultures scored at day 7 ofincubation and data points represent mean colony counts from replicate cultures.

colonies were small and of three major types-multicentric colonies containing blast cells, compact colonies usually containing immature granulocytes, and colonies of mature granulocytic cells similar to those stimulated by G-CSF. The cultures also contained a few small macrophage or granulocyte-macrophage colonies but no eosinophil, megakaryocytic, erythroid, or multipotential colonies (Table 1). SCF-stimulated colony formation was linear with respect to cultured cell numbers (Fig. 2) but, as cultured cell numbers were increased, there was a progressive rise in mean colony cell numbers (up to 10-fold), a phenomenon seen also in G-CSF-stimulated cultures. The increased colony size involved both blast cell and immature granulocytic colonies. No change occurred in the relative frequencies of the various colony types but, in cultures of high cell numbers, a few small megakaryocytic colonies were sometimes present. In cultures containing 100 ng of SCF per ml combined with 1000 units of colony-stimulating factors per ml (a 4-fold supramaximal concentration), there were only additive or subadditive effects on total colony numbers. However, with G-CSF, GM-CSF, or Multi-CSF there was a marked superadditive effect on colony size that was most evident with the combination of SCF and G-CSF (Fig. 3). In cultures with SCF and G-CSF, there was a >10-fold increase in mean colony cell numbers. The sizes of blast cell and immature granulocytic colonies were enhanced, but the cultures still contained small mature granulocytic colonies typical of those seen in cultures stimulated by either SCF or G-CSF alone. Differential colony counts suggested that combination of the two stimuli resulted in conversion of some blast colonies to granulocyte-containing colonies (Table 1). Combination of G-CSF with increasing concentrations of SCF indicated a simple additive effect on colony numbers, Table 1. Colony formation stimulated by SCF alone or in combination with G-CSF Mean Number of colonies cells per Mean Blast G GM M Stimulus per ml colonies colony 51 8 3 370 19 81 SCF, 100 ng 6 31 6 300 0 43 G-CSF, 2 ng SCF, 100 ng + 10 16 6 121 2740 89 G-CSF, 2 ng Cultures contained 75,000 C57BL/6/WEHI bone marrow cells and were analyzed on day 7 of incubation. The final concentration of stimulus in culture is indicated. Data are from duplicate cultures, and the frequency of various colonies was determined from stained preparations. Mean colony size was determined from pools of 50 sequentially sampled colonies from replicate cultures. G, granulocyte; GM, granulocyte-macrophage; M, macrophage.

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with size enhancement increasing progressively with increasing SCF concentrations (Fig. 4). A similar result was observed with the reverse combination using increasing concentrations of G-CSF. Direct Prolfferative Actions of SCF. The linearity of colony formation with cultured cell numbers suggested a direct action of SCF, but further evidence for this was sought by clone transfer studies and the culture of FACS-enriched stem and progenitor cells. The subsequent proliferation of individual day 3 clones, initiated by either SCF or G-CSF, was analyzed and then the clones were transferred to cell-free recipient cultures containing no stimulus, SCF, or G-CSF. Clones transferred were the largest 20 from each donor culture and excluded those already showing evidence of maturation from their dispersed

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FIG. 3. Effects of 103 units of CSFs or 100 ng of SCF per ml on the number and mean size (right column) of colonies developing in cultures of 75,000 C57BL/6/WEHI bone marrow cells. While combination of SCF with CSFs results in subadditive or additive colony numbers, mean colony size is increased, particularly with G-CSF. Mean data are from duplicate cultures and pools of 50 sequential colonies.

Proc. Natl. Acad. Sci. USA 88 (1991)


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FIG. 4. When 101 units of G-CSF are added to increasing concentrations of SCF, the rise in colony numbers is merely additive, but a progressive rise is observed in mean colony cell size in cultures of 75,000 C57BL/6/WEHI bone marrow cells. Curves labeled "+ saline" represent colony formation in cultures containing SCF + 0.1 ml of saline. Mean data are from duplicate cultures and pools of 50 colonies.

morphology. Most SCF-initiated clones (mean size, 20 cells) failed to survive or proliferate further when transferred to cultures containing no stimulus (Fig. 5), but many exhibited significant further proliferation in cultures containing SCF, representing evidence for a direct action of SCF. In the converse experiment, most G-CSF-initiated clones (mean size, 40 cells) failed to survive on transfer to cultures containing no stimulus, although as previously noted (9), some colony granulocytes were able to exhibit two to four further cell divisions in unstimulated cultures. Transfer of G-CSFinitiated clones to SCF-containing cultures did not result in any significant enhancement of this proliferation but continued proliferation of such clones was observed after transfer to G-CSF-containing cultures. A striking difference in this pattern was seen when SCF-initiated clones were transferred to G-CSF-containing cultures. Here, the proliferation of many clones was strongly stimulated, with the formation of granulocytic colonies containing up to 2000 cells. The data indicated that SCF does have a direct proliferative action on hemopoietic clones and that G-CSF can have a remarkable enhancing action on some SCF-initiated clones. This enhancement was unidirectional since the proliferation of G-CSF-initiated clones was not enhanced by SCF. Evidence of the ability of SCF to have direct proliferative actions on some primitive precursor cells was obtained from the culture of 200 FACS-enriched stem and progenitor cells. As reported (3), few such cells were stimulated to proliferate by SCF alone (0.3 + 0.6 of 200 cells). Nevertheless some colonies developed, including small blast cell colonies. In these cultures, G-CSF alone also stimulated little colony formation (0.3 ± 0.6 of 200 cells), but combination of the two stimuli resulted in the formation of 21.7 ± 2.5 colonies per 200 cells.

Clonogenic Cell Content of SCF-Stimulated Colonies. Sequential colonies stimulated by 100 ng of SCF per ml, 103

FIG. 5. Size achieved by intact day 3 clones initiated by 100 ng of SCF or 103 units of G-CSF 4 days after transfer to cell-free recipient cultures containing no stimulus (0), 100 ng of SCF, or 103 units of G-CSF. The mean size of clones at transfer is indicated by the horizontal lines.

units of G-CSF per ml, or both were resuspended and then recultured in cultures stimulated by G-CSF, SCF, or 0.1 ml of pokeweed mitogen-stimulated spleen cell-conditioned medium (SCM) (6) [SCM constitutes a combined stimulus containing GM-CSF, Multi-CSF, and interleukins 4, 5, and 6 (IL-4, IL-5, and IL-6]. No G-CSF-stimulated colonies contained clonogenic cells detectable with any of the three stimuli used in secondary cultures. In SCM-stimulated secondary cultures, clonogenic cells were present in only 10 of 35 SCF-stimulated granulocytic colonies (16 + 16 per colony) and in only 16 of 41 compact SCF-stimulated colonies (30 + 49 per colony). In contrast, 33 of 40 SCF-stimulated multicentric blast colonies contained progenitor cells detectable in SCM-stimulated cultures (72 + 79 per colony). In SCFstimulated secondary cultures, 21 of 40 SCF-initiated blast cell colonies contained progenitor cells (16 + 10 per colony). With a mean size of 300 cells for SCF-stimulated blast cell colonies, the data indicated that an average of 25% of the colony cells must have been progenitor cells, and in a few colonies the frequency approached 100%6. In no instance did blast colonies develop, even in SCF-containing secondary cultures; with SCM as the stimulus, 44% of secondary colonies were macrophage, 12% granulocyte-macrophage and 44% granulocytic in composition. SCF-stimulated secondary colonies were exclusively composed of granulocytes. The same pattern was observed after recloning of colonies stimulated by a mixture of SCF and G-CSF. The highest frequency of progenitor cells was observed in multicentric blast colonies (mean size, -3000 cells), and in recipient cultures containing SCM, 22 of 23 colonies contained 532 + 389 progenitor cells per colony, the secondary colonies again mainly containing macrophages and/or granulocytes. In secondary cultures containing SCF or G-CSF, smaller numbers of progenitor cells were detected, and these exclusively formed granulocytic colonies. The mean number of clonogenic cells in the enlarged blast colonies stimulated by the

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Table 2. 1251-SCF labeling of adult BALB/c mouse bone marrow cells Labeled cells % of Mean grain total Cell type count % label Blast cells 86 130 65 Promyelocytes/myelocytes 75 9 3 Metamyelocytes/polymorphs 4 3 1 Promonocytes 84 21 10 Monocytes 51 7 3 Eosinophilic myelocytes 100 20 4 Eosinophils 45 6 1 Lymphocytes 7 47 13 Nucleated erythroid cells 0 0 0 Autoradiograph exposure time was 21 days. Data represent background-subtracted grain counts over 50-100 sequential cells of each type. Percent contribution to total labeling was calculated from the differential count of the BALB/c marrow cells used.

combination of G-CSF with SCF was increased 7-fold compared with that in SCF-stimulated blast cell colonies. Again, no blast cell colonies developed in secondary cultures stimulated by SCM, SCF, or G-CSF. Distribution of SCF Receptors on Marrow Ceils. Autoradiographic analysis of normal BALB/c marrow cells preincubated with 1251-SCF showed prominent labeled cells, and this labeling was blocked completely by coincubation with a 40-fold excess of unlabeled SCF. As shown in Table 2, the pattern of labeling was highly distinctive. Most blast cells were labeled and exhibited an unusually high number of grains per cell. In the granulocytic series, most promyelocytes and myelocytes were also labeled, but grain counts were very low; few metamyelocytes and no polymorphs were labeled. Monocytic cells were usually more heavily labeled than granulocytic cells of comparable maturation stage, and this was reflected in the labeling of =50% of mature monocytes, although the grain counts on these cells were low. Eosinophilic myelocytes and mature eosinophils exhibited the same labeling pattern as monocytic cells. No labeling was observed of nucleated erythroid cells. Most lymphocytes exhibited no labeling, but 7% of the cells were labeled, often with the same high grain counts as observed on blast cells. These cells had the morphology of small or medium lymphocytes. Despite their low frequency (4% of the total population), the blast cells accounted for two-thirds of the total binding of 1251-SCF to marrow cells.

DISCUSSION SCF was purified on the basis of its capacity to stimulate the formation of hemopoietic colonies by primitive hemopoietic precursors from bone marrow and the proliferation of mast cell lines (3, 4). Because ofthe striking synergy evident in this proliferation of marrow cells when colony-stimulating factors or IL-6 was present, SCF has been regarded as possibly being mainly an enhancing factor, amplifying the action of other growth factors. The present experiments were designed to establish (i) whether or not SCF is a direct proliferative stimulus for normal murine marrow cells and (ii) the nature of the enhancement evident when SCF is used in combination with the CSFs. This latter study concentrated on the SCF-G-CSF combination, since this was quantitatively the most striking and involved a CSF with an action relatively restricted to granulocyte precursor cells (6). The data indicate strongly that SCF is a direct-acting proliferative stimulus inducing the formation of multicentric blast cell colonies and immature or maturing granulocytic colonies. The evidence supporting this conclusion is (i) the

Proc. Nad. Acad. Sci. USA 88 (1991) presence of large numbers of SCF receptors on blast cells (the morphological population containing progenitor cells), (it) the linearity of SCF-induced colony formation with varying numbers of cultured cells, (iii) the ability of SCF-initiated clones to continue proliferation after transfer to cultures containing SCF but no other cells, (iv) the ability of SCF to

stimulate some colony formation by recultured 7-day colony cells, and (v) the ability of SCF to stimulate some colony formation by enriched populations freed of accessory cells. These are the same criteria used previously to establish direct proliferative actions of other hemopoietic regulators (6). While the data indicate a direct proliferative action of SCF, 1000-fold higher concentrations of SCF are required than with the CSFs. This is curious in view of the high receptor numbers for SCF on blast cells-the population likely to contain the clonogenic cells responding to SCF. The development of blast cell colonies after SCF stimulation is highly distinctive and might suggest a capacity of SCF to induce self-generation by progenitor cells. However, despite the high content of progenitor cells in blast colonies, no blast colony-forming cells were detected. Therefore, these data provide no evidence for a capacity of SCF to stimulate self-generation by blast colony-forming cells. Because many of the progenitor cells in blast colonies appear to be macrophage precursors and SCF receptors are present on monocyte-macrophage populations, it is curious why so few macrophage-containing colonies develop in SCF-stimulated primary cultures. Equally curious is the ability of SCF to stimulate the formation of mature granulocytic colonies when receptors are not detectable on metamyelocytes and polymorphs. Combination of SCF with G-CSF resulted in merely an additive effect on colony numbers, suggesting some independence of action, perhaps based on the existence of differing subsets of progenitor cells able to respond initially to each stimulus. However, combination of SCF with G-CSF must also result in a synergistic interaction on many individual clones, since these exhibited a major increase in their size and content of progenitor cells. This synergistic interaction appeared to be restricted to clonogenic cells able to be initiated by SCF, since transfer of G-CSF-initiated clones to SCF did not result in a significant increase in their further proliferation. In the presence of SCF, G-CSF is able to strongly amplify progenitor cell generation and the formation of large numbers of maturing granulocyte progeny-actions not observable in cultures stimulated by G-CSF alone. This phenomenon may help to explain the obvious quantitative discrepancy that exists between the weak proliferative action of G-CSF acting alone in vitro (6), and the effects of G-CSF in vivo, where major rises in neutrophil levels are readily inducible by G-CSF injection (10, 11). It is of interest that G-CSF has only a very weak capacity to elevate neutrophil levels in W and Steel mutant mice (12), suggesting that the strong response elicited by G-CSF in normal mice and most humans may depend on an enhancement interaction with SCF. We thank Dr. K. Zsebo and Dr. L. Souza of Amgen (Thousand Oaks, CA) for generously supplying, respectively, the SCF and G-CSF used in these studies; Mrs. L. Di Rago and Miss S. Mifsud for technical assistance; and Dr. C. Li for supplying the FACSpurified mouse bone marrow stem cells used. This work was supported by the Carden Fellowship Fund of the Anti-Cancer Council of Victoria; the National Health and Medical Research Council, Canberra, and the National Institutes of Health Grant 22556. 1. Charbot, B., Stephenson, D. A., Chapman, V. M., Besmer, P. & Bernstein, A. (1988) Nature (London) 335, 88-89. 2. Geissler, E. N., Ryan, M. A. & Housman, D. E. (1988) Cell 55, 185-192. 3. Zsebo, K. M., Wypych, J., McNiece, I. K., Lu, H. S., Smith,

Medical Sciences: Metcalf and Nicola K. A., Karkare, S. B., Sachdev, R. K., Yuschenkoff, V. N., Birkett, N. C., Williams, L. R., Satyagal, V. N., Tung, W., Bosselman, R. A., Mendiaz, E. A. & Langley, K. E. (1990) Cell 63, 195-201. 4. Anderson, K. M., Lyman, S. D., Baird, A., Wignall, J. M., Eisenman, J., Rauch, C., March, C. J., Boswell, H. S., Gimpel, S. D., Cosman, D. & Williams, D. E. (1990) Cell 63, 235-243. 5. Martin, F. H., Suggs, S. V., Langley, K. E., Lu, H. S., Ting, J., Okino, K. H., Morris, C. F., McNiece, I. K., Jacobsen, F. W., Mendiaz, E. A., Birkett, N. C., Smith, J. A., Johnson, M. J., Parker, V. P., Flores, J. C., Patel, A. C., Fisher, E. F., Eijavec, H. O., Herrera, C. J., Wypych, J., Sachdev, R. K., Pope, J. A., Leslie, I., Wen, D., Len, C.-H., Cupples, R. L. & Zsebo, K. M. (1990) Cell 63, 203-211.

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